Systems and methods for identifying a culture as positive for microorganisms with high confidence

ABSTRACT

Systems, methods, and apparatus for determining whether a culture in a vessel contains a plurality of microorganisms are provided. A normalization relative value is calculated for each respective measurement of a biological state of the culture between (i) the respective measurement and (ii) an initial biological state. For each fixed interval of time points, a derivative of the normalization relative values in the interval of time points is calculated, thereby forming a plurality of rate transformation values. For each set of rate transformation values in the plurality of rate transformation values, a measure of central tendency of the values in the set is computed, thereby forming a plurality of average relative transformation values. A determination whether the culture contains the microorganisms is made based on whether any calculated average relative transformation value exceeds a first threshold or whether an extent of growth exhibited by the culture exceeds a second threshold.

1 FIELD OF THE INVENTION

Disclosed are improved systems and methods for determining that aculture in a vessel contains microorganisms.

2 BACKGROUND OF THE INVENTION

Rapid and reliable detection of microorganisms in a culture, such as ablood culture, is among the most important functions of the clinicalmicrobiology laboratory. Currently, the presence of biologically activeagents such as bacteria in a patient's body fluid, and especially inblood, is determined using culture vials. A small quantity of thepatient's body fluid is injected through an enclosing rubber septum intoa sterile vial containing a culture medium and the vial is thenincubated and monitored for microorganism growth.

Common visual inspection of the culture vial then involves monitoringthe turbidity or observing eventual color changes of the liquidsuspension within the vial. Known instrument methods can also be used todetect changes in the carbon dioxide content of the culture vessels,which is a metabolic byproduct of the bacterial growth. Monitoring thecarbon dioxide content can be accomplished by methods well establishedin the art.

In some instances, non-invasive infrared microorganism detectioninstrument is used in which special vials having infrared-transmittingwindows are utilized. In some instances, glass vials are transferred toan infrared spectrometer by an automated manipulator arm and measuredthrough the glass vial. In some instances, chemical sensors are disposedinside the vial. These sensors respond to changes in the carbon dioxideconcentration in the liquid phase by changing their color or by changingtheir fluorescence intensity. These techniques are based on lightintensity measurements and require spectral filtering in the excitationand/or emission signals.

As the above indicates, several different culture systems and approachesare available to laboratories. For example, the BACTEC® radiometric andnonradiometric systems (Becton Dickenson Diagnostic Instrument Systems,Sparks, Md.) are often used for this task. The BACTEC® 9240 instrument,for example, accommodates up to 240 culture vessels and serves as anincubator, agitator, and detection system. Each vessel contains afluorescent CO₂ sensor, and the sensors are monitored on a continuousbasis (e.g., every ten minutes). Cultures are recognized as positive bycomputer algorithms for growth detection based on an increasing rate ofchange as well as sustained increase in CO₂ production rather than bythe use of growth index threshold or delta values. The BACTEC® 9240 iscompletely automated once the vessels have been loaded.

One drawback with these microorganism detection approaches is that theydo not always detect cultures that contain microorganisms. Thus, whatare needed in each of the above-identified systems are improved methodsfor determining whether a culture in a vessel contains a plurality ofmicroorganisms.

3 SUMMARY OF THE INVENTION

To meet the needs identified in the prior art, the present invention, inone aspect, provides systems, methods and apparatus that allow anincreased confidence level in the notification of vessel positive statusin culture systems. The present invention advantageously provides a highconfidence positive status in a blood culture.

The present invention utilizes the difference in rate of metabolicchange and extent of change to provide information about the confidencein a positive status change on an individual vessel basis. the presentinvention describes a data transformation that can be applied tometabolic or cell growth data in a way that provides confidence that aculture in a vessel is infected with a microorganism (high confidencepositive) and essentially eliminates the potential for false negativedeterminations as they currently exist in known culture systems. Thehigh confidence positive can, for example, be applied to cases whengrowth has begun but the vial was not being measured. An example is thecase when a vessel encounters significant delays between the time thespecimen was collected into the vessel and the time the vessel entersthe measuring instrument. The high confidence positive algorithm can beapplied to vessels that have measurement reading gaps resulting from anumber of causes including loss of power, instrument failure and downtime due to service. The user benefit is a decreased requirement tosubculture vessels that have encountered these types of protocolinterruptions. Further, the high confidence positive may be linked topositive test procedures as a biological quantification metric. Forexample, a culture may be detected as positive at an average rate changevalue (ART) value of 100, the cell mass of an ART=200 may be required toperform a rapid identification or molecular characterization of themicroorganism present, and an ART value >400 may require dilution priorto rapid identification or gene typing procedures.

In one aspect, the present invention provides a method of determiningwhether a culture in a vessel contains a plurality of microorganisms. Inthe method a normalization relative value is calculated for eachrespective measurement in a plurality of measurements of a biologicalstate of the culture in the vessel, taken at different time pointsbetween a first time point and a second time point, between (i) therespective measurement and (ii) an initial biological state of theculture taken at an initial time point, thereby forming a plurality ofnormalization relative values.

The plurality of normalization relative values can be broken down, on atimewise basis, into predetermined fixed intervals of time pointsbetween the first time point and the second time point. For instance, afirst predetermined fixed interval may include the first tennormalization relative values, a second predetermined fixed interval mayinclude the next ten normalization relative values, and so forth untilthe second time point is reached. For each of these respectivepredetermined fixed intervals of time points between the first timepoint and the second time point, a first derivative of the normalizationrelative values in the respective predetermined fixed interval isdetermined, thereby forming a plurality of rate transformation values.

There is a rate transformation value for each predetermined fixedinterval of time points. The plurality of rate transformation values canbe considered as comprising a plurality of sets of rate transformationvalues. Each respective set of rate transformation values is for adifferent set of contiguous time points between the first time point andthe second time point. For example, the first set of rate transformationvalues may be the first seven rate transformation values in theplurality of rate transformation values, the second set of ratetransformation values may be the next seven rate transformation valuesin the plurality of rate transformation values, and so forth. For eachrespective set of rate transformation values in the plurality of sets ofrate transformation values, an average relative transformation value iscomputed as a measure of central tendency of each of the ratetransformation values in the respective set of rate transformationvalues. In this way, a plurality of average relative transformationvalues is computed.

Further, in the method, either (i) a first result, (ii) a second result,or (iii) both a first or second result is obtained. The first result isbased on a determination of whether any average relative transformationvalue in the plurality of average relative transformation values exceedsa first threshold value. The second result is based on a determinationof whether an extent of growth exhibited by the culture exceeds a secondthreshold value. The first result or the second result is used todetermine whether the culture in the vessel contains the plurality ofmicroorganisms.

In some embodiments, the method further comprises outputting the firstresult, the second result, or a determination of whether the culture inthe vessel contains the plurality of microorganisms to a user interfacedevice, a monitor, a computer-readable storage medium, acomputer-readable memory, or a local or remote computer system. In someembodiments, the first result, the second result, or the determinationof whether the culture in the vessel contains the plurality ofmicroorganisms is displayed.

In some embodiments, the first time point is five or more minutes afterthe initial time point and the final time point is thirty or more hoursafter the initial time point. In some embodiments, the first time pointis between 0.5 hours and 3 hours after the initial time point and thefinal time point is between 4.5 hours and twenty hours after the initialtime point. In some embodiments, the measure of central tendency of therate transformation values in a first set of rate transformation valuesin the plurality of sets of rate transformation values comprises (i) ageometric mean, an arithmetic mean, a median, or a mode of each of therate transformation values in the first set of rate transformationvalues.

In some embodiments, the measurements in the plurality of measurementsof the biological state of the culture are each taken of the culture ata periodic time interval between the first time point and the secondtime point. In some embodiments, the periodic time interval is an amountof time between one minute and twenty minutes, between five minutes andfifteen minutes, or between 0.5 minutes and 120 minutes.

In some embodiments, the initial biological state of the culture isdetermined by a fluorescence output of a sensor that is in contact withthe culture. For instance, in some embodiments, the amount offluorescence output of the sensor is affected by CO₂ concentration, O₂concentration, or pH.

In some embodiments, between 10 and 50,000 measurements, between 100 and10,000 measurements, or between 150 and 5,000 measurements of thebiological state of the culture in the vessel are in the plurality ofmeasurements of the biological state of the culture. In someembodiments, each respective predetermined fixed interval of time pointscomprises or consists of each of the rate transformation values for timepoints in a time window between the first time point and the second timepoint, where the time window is a period of time that is between twentyminutes and ten hours, twenty minutes and two hours, or thirty minutesand ninety minutes.

In some embodiments, each set of rate transformation values in theplurality of rate transformation values comprises or consists of betweenfour and twenty, between five and fifteen, or between 2 and 100contiguous rate transformation values. In some embodiments, there arebetween five and five hundred or between twenty and one hundred averagerelative transformation values in the plurality of average relativetransformation values. In some embodiments, a volume of the culture isbetween 1 ml and 40 ml, between 2 ml and 10 ml, less than 100 ml, orgreater than 100 ml.

In some embodiments, the vessel contains a sensor composition in fluidcommunication with the culture, where the sensor composition comprises aluminescent compound that exhibits a change in luminescent property,when irradiated with light containing wavelengths that cause saidluminescent compound to luminesce, upon exposure to oxygen, and wherethe presence of the sensor composition is non-destructive to the cultureand where the initial biological state of the culture is measured by themethod of (i) irradiating the sensor composition with light containingwavelengths that cause the luminescent compound to luminesce and (ii)observing the luminescent light intensity from the luminescent compoundwhile irradiating the sensor composition with the light. In someembodiments, the luminescent compound is contained within a matrix thatis relatively impermeable to water and non-gaseous solutes, but whichhas a high permeability to oxygen. In some embodiments, the matrixcomprises rubber or plastic.

In some embodiments, the extent of growth (EG) is the maximumnormalization relative value in the plurality of normalization relativevalues. In some embodiments, the extent of growth is determined by theequation:

EG=NR_(after_growth)−NR_(minimum_growth)

where

NR_(after_growth) is a normalization relative value in the plurality ofnormalization relative values that was used in the calculation of (i)the first average relative transformation value following a maximumaverage relative transformation value, (ii) a maximum average relativetransformation value, or (iii) a first average relative transformationvalue preceding the maximum average relative transformation value in theplurality of average relative transformation values, and

NR_(minimum_growth) is a normalization relative value in the pluralityof normalization relative values that was used in the calculation of thefirst average relative transformation value to achieve a third thresholdvalue. In some embodiments, the third threshold value is a value between5 and 100 or a value between 25 and 75.

Advantageously, using the novel systems, methods, and apparatus of thepresent invention, an incubating system, such as the BACTEC® bloodculture system, can be programmed to determine whether a culture isinfected with microorganisms before manual tests, such as a Gram stainor a subculture, are performed. Briefly, a culture is identified aspositive for microorganism infection by an incubator by analyzing novelparameters (e.g., average relative transformation value, extent ofgrowth exhibited by the culture) associated with microorganismmetabolism. Such cultures will have increased metabolism relative touninfected cultures and, on this basis, microorganism infection can bedetected. While the tests disclosed herein are most accurate when asingle microorganism type is infecting a culture, it is possible todetect microorganism infection when multiple microorganism types (e.g.,multiple microorganism species) infect a single culture.

While numerous exemplary values for novel parameters (e.g., averagerelative transformation value, extent of growth exhibited by theculture) disclosed herein are given in the data presented herein fordetecting whether a culture is infected with microorganisms using agiven media, it is to be appreciated that these values for the novelparameters may change when the media used to support growth of theculture is altered. Moreover, it is possible that the values of thenovel parameters may vary when a different incubator is used. Thus,preferentially, the same incubator used to generate reference values forthe detection of microorganism infection should be used for cultureswhere the microorganism status is not known. Moreover, the same culturemedia used to generate reference values for the detection ofmicroorganism infection should be used for cultures where themicroorganism status is not known.

In some embodiments, the culture in the vessel is deemed to contain theplurality of microorganisms when an average relative transformationvalue in the plurality of average relative transformation values exceedsthe first threshold value. In some embodiments, the culture in thevessel is deemed to contain the plurality of microorganisms when theextent of growth exhibited by the culture exceeds the second thresholdvalue.

In some embodiments, the method determines that the culture in thevessel contains the plurality of microorganisms and the plurality ofmicroorganisms is bacteria in the Enterobacteriaceae family. In someembodiments, the method determines that the culture contains theplurality of microorganisms, and the plurality of microorganisms in theculture is (i) Enterobacteriaceae, (ii) Staphylococcaceae, (iii)Streptococcus, or (iv) Acinetobacter. In some embodiments, the methoddetermines that the culture contains the plurality of microorganisms andthe plurality of microorganisms are Alishewanella, Alterococcus,Aquamonas, Aranicola, Arsenophonus, Azotivirga, Blochmannia, Brenneria,Buchnera, Budvicia, Buttiauxella, Cedecea, Citrobacter, Dickeya,Edwardsiella, Enterobacter, Erwinia, Escherichia, Ewingella,Griimontella, Hafnia, Klebsiella, Kluyvera, Leclercia, Leminorella,Moellerella, Morganella, Obesumbacterium, Pantoea, Pectobacterium,Candidatus phlomobacter, Photorhabdus, Plesiomonas, Pragia, Proteus,Providencia, Rahnella, Raoultella, Salmonella, Samsonia, Serratia,Shigella, Sodalis, Tatumella, Trabulsiella, Wigglesworthia, Xenorhabdus,Yersinia, or Yokenella.

In some embodiments, the method determines that the culture in thevessel contains the plurality of microorganisms and the plurality ofmicroorganisms are a single species of Staphylococcaceae selected fromthe group consisting of Staphylococcus aureus, Staphylococcus caprae,Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcushominis, Staphylococcus lugdunensis, Staphylococcus pettenkoferi,Staphylococcus saprophyticus, Staphylococcus warneri, and Staphylococcusxylosus.

In some embodiments, the method determines that the culture contains theplurality of microorganisms and the plurality of microorganisms isStaphylococcus aureus or coagulase negative staphylococci. In someembodiments the method determines that the culture contains theplurality of microorganisms and the plurality of microorganisms are asingle species of Streptococcus selected from the group consisting of S.agalactiae, S. bovis, S. mutans, S. pneumoniae, S. pyogenes, S.salivarius, S. sanguinis, S. suis, Streptococcus viridans, andStreptococcus uberis.

In some embodiments, the method determines that the culture in thevessel contains the plurality of microorganisms and the plurality ofmicroorganisms is aerobic. In some embodiments, the method determinesthat the culture in the vessel contains the plurality of microorganisms,and the plurality of microorganisms is anaerobic. In some embodiments,the initial biological state of the culture is measured by acolorimetric means, a fluorometric means, a nephelometric means, or aninfrared means. In some embodiments, each biological state in theplurality of measurements of the biological state is determined by acolorimetric means, a fluorometric means, a nephelometric means, or aninfrared means. In some embodiments, the culture is a blood culture froma subject.

In some embodiments, only the first result is obtained and used todetermine whether the culture in the vessel contains the plurality oforganisms. In some embodiments, only the second result is used todetermine whether the culture in the vessel contains the plurality oforganisms. In some embodiments, the first result and the second resultare used to determine whether the culture in the vessel contains theplurality of organisms.

In another aspect, the present invention provides an apparatus fordetermining whether a culture in a vessel contains a plurality ofmicroorganisms in which the apparatus comprises a processor and amemory, coupled to the processor, for carrying out any of the methodsdisclosed herein. In still another aspect of the present invention, acomputer-readable medium storing a computer program product fordetermining whether a culture in a vessel contains a plurality ofmicroorganisms, where the computer program product is executable by acomputer. The computer program product comprises instructions forcarrying out any of the methods disclosed herein.

In another aspect, the present invention provides a method ofdetermining whether a culture in a vessel contains a plurality ofmicroorganisms. The method comprises obtaining a plurality ofmeasurements of the biological state of the culture, each measurement inthe plurality of measurements taken at a different time point between afirst time point and a second time point. The method further comprisesdetermining, for each respective predetermined fixed interval of timepoints between the first time point and the second time point, a firstderivative of the measurements of the biological state in the respectivepredetermined fixed interval of time points, thereby forming a pluralityof rate transformation values, where the plurality of ratetransformation values comprises a plurality of sets of ratetransformation values, where each respective set of rate transformationvalues in the plurality of sets of rate transformation values is for adifferent set of contiguous time points between the first time point andthe second time point. The method further comprises computing, for eachrespective set of rate transformation values in the plurality of sets ofrate transformation values, an average relative transformation value asa measure of central tendency of each of the rate transformation valuesin the respective set of rate transformation values, thereby computing aplurality of average relative transformation values. The method furthercomprises obtaining (i) a first result based on a determination ofwhether any average relative transformation value in the plurality ofaverage relative transformation values exceeds a first threshold valueor (ii) a second result based on a determination of whether an extent ofgrowth exhibited by the culture exceeds a second threshold value. Themethod further comprises using the first result or the second result todetermine whether the culture in the vessel contains the plurality ofmicroorganisms.

As such, the systems and methods of the present invention can provide anumber of applications useful in microbiology and related fields, andfinds particular application in cell culture sterility test procedures.

4 BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an apparatus for determining whether a culture in avessel contains a plurality of microorganisms, the apparatus comprisinga processor and a memory, coupled to the processor, in accordance withan embodiment of the present invention.

FIG. 2 illustrates a schematic drawing of a culture vessel and CO₂detector system in accordance with an embodiment of the presentinvention.

FIGS. 3A & 3B illustrate a method for determining whether a culture in avessel contains a plurality of microorganisms in accordance with anembodiment of the present invention.

FIG. 4 shows a plot of normalization relative values measured from ablood culture in a vessel in accordance with an embodiment of thepresent invention.

FIG. 5 is a plot of the average relative transformation values over timebased on the average rate of change in rate transformation values ofFIG. 4 over time in accordance with an embodiment of the presentinvention.

FIG. 6 is the second derivative plot of normalization relative values ofFIG. 4 and shows the changes in metabolism rate with time in accordancewith an embodiment of the present invention.

FIG. 7 is a plot of compensated fluorescent signal versus time for aclinical Enterococcus faecalis false negative.

FIG. 8 is a plot of normalized relative values versus time for aclinical Enterococcus faecalis false negative.

FIG. 9 is a plot of average rate transformation versus time for aclinical Enterococcus faecalis false negative.

Like reference numerals refer to corresponding parts throughout theseveral views of the drawings.

5 DETAILED DESCRIPTION OF THE INVENTION

Systems, methods, and apparatus for determining whether a culture in avessel contains a plurality of microorganisms are provided. Anormalization relative value is calculated for each respectivemeasurement of a biological state of the culture between (i) therespective measurement and (ii) an initial biological state. For eachfixed interval of time points between a first time point and a secondtime point, a derivative of the normalization relative values formeasurements of the biological state in the interval of time points iscalculated, thereby forming a plurality of rate transformation values.For each set of rate transformation values in the plurality of ratetransformation values, a measure of central tendency of the values inthe set is computed, thereby forming a plurality of average relativetransformation values. A determination of whether the culture containsthe microorganisms is made based on whether any calculated averagerelative transformation value exceeds a first threshold or whether anextent of growth exhibited by the culture exceeds a second threshold.

One such system in which the present invention can be implemented is theBACTEC® blood culture system. The BACTEC® blood culture system usesfluorescent sensors that report changes to the system when microbialmetabolism occurs. Algorithms are then applied to the sequence of signaldata that are designed to recognize signal changes with time that areindicative of the presence of growing microorganisms. The user isnotified when the system recognizes evidence of growth (status change toa positive vial) and the vessel is then processed to confirm thepresence of a microorganism (e.g. using a gram stain and subculture to aplated medium) before initiating processes to begin organismidentification and antimicrobial susceptibility determinations.

5.1 Definitions

The term “Acinetobacter” as used herein refers to a Gram-negative genusof bacteria belonging to the phylum Proteobacteria. Non-motile,Acinetobacter species are oxidase-negative, and occur in pairs undermagnification.

The term “biological state” as used herein refers to a measure of themetabolic activity of a culture as determined by, for example, CO₂concentration, O₂ concentration, pH, a rate of change in CO₂concentration, a rate of change in O₂ concentration, or a rate of changein pH in the culture.

The term “blood” as used herein means either whole blood or any one,two, three, four, five, six, or seven cell types from the group of cellstypes consisting of red blood cells, platelets, neutrophils,eosinophils, basophils, lymphocytes, and monocytes. Blood can be fromany species including, but not limited to, humans, any laboratory animal(e.g., rat, mouse, dog, chimp), or any mammal.

The term “blood culture” as used herein refers to any amount of bloodthat has been mixed with blood culture media. Examples of culture mediainclude, but are not limited to, supplemented soybean casein broth,soybean casein digest, hemin, menadione, sodium bicarbonate, sodiumpolyaneltholesulfonate, sucrose, pyridoxal HCKl, yeast extract, andL-cysteine. One or more reagents that may be used as blood culture mediaare found, for example, in Stanier et al., 1986, The Microbial World,5^(th) edition, Prentice-Hall, Englewood Cliffs, N.J., pages 10-20,33-37, and 190-195, which is hereby incorporated by reference herein inits entirety for such purpose. In some instances, a blood culture isobtained when a subject has symptoms of a blood infection or bacteremia.Blood is drawn from a subject and put directly into a vessel containinga nutritional culture media. In some embodiments, ten milliliters ofblood is needed for each vessel.

The term “culture” as used herein refers to any biological sample from asubject that is either in isolation or mixed with one or more reagentsthat are designed to culture cells. The biological sample from thesubject can be, for example, blood, cells, a cellular extract, cerebralspinal fluid, plasma, serum, saliva, sputum, a tissue specimen, a tissuebiopsy, urine, a wound secretion, a sample from an in-dwelling linecatheter surface, or a stool specimen. The subject can be a member ofany species including, but not limited to, humans, any laboratory animal(e.g., rat, mouse, dog, chimp), or any mammal. One or more reagents thatmay be mixed with the biological sample are found, for example, inStanier et al., 1986, The Microbial World, 5^(th) edition,Prentice-Hall, Englewood Cliffs, N.J., pages 10-20, 33-37, and 190-195,which is hereby incorporated by reference herein in its entirety forsuch purpose. A blood culture is an example of a culture. In someembodiments, the biological sample is in liquid form and the amount ofthe biological sample in the culture is between 1 ml and 150 ml, between2 ml and 100 ml, between 0.5 ml and 90 ml, between 0.5 ml and 10,000 ml,or between 0.25 ml and 100,000 ml. In some embodiments, the biologicalsample is in liquid form and is between 1 and 99 percent of the volumeof the culture, between 5 and 80 percent of the volume of the culture,between 10 and 75 percent of the volume of the culture, less than 80percent of the volume of the culture, or greater than 10 percent of thevolume of the culture. In some embodiments, the biological sample isbetween 1 and 99 percent of the total weight of the culture, between 5and 80 percent of the total weight of the culture, between 10 and 75percent of the total weight of the culture, less than 80 percent of thetotal weight of the culture, or greater than 10 percent of the totalweight of the culture.

As used herein, the term “Enterobacteriaceae” refers to a large familyof bacteria, including Salmonella and Escherichia coli.Enterobacteriaceae are also referred to herein as the Enteric group.Genetic studies place them among the Proteobacteria, and they are giventheir own order (Enterobacteriales). Members of the Enterobacteriaceaeare rod-shaped, and are typically 1 μm to 5 μm in length. Like otherproteobacteria they have Gram-negative stains, and they are facultativeanaerobes, fermenting sugars to produce lactic acid and various otherend products. They also reduce nitrate to nitrite. Unlike most similarbacteria, Enterobacteriaceae generally lack cytochrome C oxidase,although there are exceptions (e.g. Plesiomonas). Most have manyflagella, but a few genera are non-motile. They are non-spore forming,and except for Shigella dysenteriae strains they are catalase-positive.Many members of this family are a normal part of the gut flora found inthe intestines of humans and other animals, while others are found inwater or soil, or are parasites on a variety of different animals andplants. Most members of Enterobacteriaceae have peritrichous Type Ifimbriae involved in the adhesion of the bacterial cells to their hosts.Genera of the Enterobacteriaceae include, but are not limited to,Alishewanella, Alterococcus, Aquamonas, Aranicola, Arsenophonus,Azotivirga, Blochmannia, Brenneria, Buchnera, Budvicia, Buttiauxella,Cedecea, Citrobacter, Dickeya, Edwardsiella, Enterobacter, Erwinia (e.g.Erwinia amylovora), Escherichia (e.g. Escherichia coli), Ewingella,Griimontella, Hafnia, Klebsiella (e.g. Klebsiella pneumoniae), Kluyvera,Leclercia, Leminorella, Moellerella, Morganella, Obesumbacterium,Pantoea, Pectobacterium, Candidatus phlomobacter, Photorhabdus (e.g.,Photorhabdus luminescens), Plesiomonas (e.g. Plesiomonas shigelloides),Pragia, Proteus (e.g. Proteus vulgaris), Providencia, Rahnella,Raoultella, Salmonella, Samsonia, Serratia (e.g. Serratia marcescens),Shigella, Sodalis, Tatumella, Trabulsiella, Wigglesworthia, Xenorhabdus,Yersinia (e.g., Yersinia pestis), and Yokenella. More information aboutEnterobacteriaceae is found in Stanier et al., 1986, The MicrobialWorld, 5^(th) edition, Prentice-Hall, Englewood Cliffs, N.J., Chapter 5,which is hereby incorporated by reference herein for such purpose.

As used herein, the term “instance” refers to the execution of a step inan algorithm. Some steps in an algorithm may be run several times, witheach repeat of the step being referred to as an instance of the step.

As used herein, the term “microorganism” refers to organisms with adiameter of 1 mm or less excluding viruses.

As used herein, the term “microorganism type” refers to anysubclassification of the bacteria kingdom such as a phylum, class,order, family, genus or species in the bacteria kingdom.

As used herein, the term “portion” refers to at least one percent, atleast two percent, at least ten percent, at least twenty percent, atleast thirty percent, at least fifty percent, as least seventy-fivepercent, at least ninety percent, or at least 99 percent of a set. Thus,in a nonlimiting example, at least a portion of a plurality of objectsmeans at least one percent, at least two percent, at least ten percent,at least twenty percent, at least thirty percent, at least fiftypercent, as least seventy-five percent, at least ninety percent, or atleast 99 percent of the objects in the plurality.

As used herein, the term “Staphylococcaceae” refers to a family ofbacteria in the Bacillales order that includes, but is not limited to,the Staphylococcus aureus, Staphylococcus caprae, Staphylococcusepidermidis, Staphylococcus haemolyticus, Staphylococcus hominis,Staphylococcus lugdunensis, Staphylococcus pettenkoferi, Staphylococcussaprophyticus, Staphylococcus warneri, and Staphylococcus xylosusbacteria.

As used herein, the term “Streptococcus” refers to a genus of sphericalGram-positive bacteria, belonging to the phylum Firmicutes and thelactic acid bacteria group. Cellular division occurs along a single axisin these bacteria, and thus they grow in chains or pairs, hence thename—from Greek streptos, meaning easily bent or twisted, like a chain.This is contrasted with staphylococci, which divide along multiple axesand generate grape-like clusters of cells. Species of Streptococcusinclude, but are not limited to S. agalactiae, S. bovis, S. mutans, S.pneumoniae, S. pyogenes, S. salivarius, S. sanguinis, S. suis,Streptococcus viridans, and Streptococcus uberis.

As used herein, a “subject” is an animal, preferably a mammal, morepreferably a non-human primate, and most preferably a human. The terms“subject”, “individual” and “patient” are used interchangeably herein.

As used herein, the term “vessel” refers to any container that can holda culture such as a blood culture. For instance, in one embodiment avessel is a container having a side wall, a bottom wall, an open top endfor receiving a culture to be contained within the container, where thecontainer is formed from a material such as glass, clear plastic (e.g.,a cyclic olefin copolymer) having a transparency sufficient to visuallyobserve turbidity in the sample, and where the is preferably resistantto heating at a temperature of at least 250° C. In some embodiments, thecontainer has a wall thickness sufficient to withstand an internalpressure of at least 25 psi and a closure coupled to the open end of thecontainer, where the culture is substantially free of contaminationafter storage in the vessel for an extended period of time under ambientconditions. Exemplary containers are described in U.S. Pat. No.6,432,697, which is hereby incorporated herein by reference. In someembodiments, the extended period of time under ambient conditions is atleast about one year at about 40° C. In some embodiments, the vesselfurther comprises a fluorescent sensor compound fixed to an innersurface of the container that, when exposed to oxygen, exhibits areduction in fluorescent intensity upon exposure to a fluorescing light.In some embodiments, the container is substantially transparent to saidfluorescing light. In some embodiments, the fluorescent sensor compoundcomprises at least one compound selected from the group consisting oftris-4,7-diphenyl-1,10-phenanthroline ruthenium (II) salts,tris-2,2′-bipyridyl ruthenium (II) salts, 9,10-diphenyl anthracene, andmixtures thereof. In some embodiments, a vessel is a Blood CultureBACTEC® LYTIC/10 Anaerobic/F culture vial, a BBL® SEPTI-CHEK® vial, aBBL® SEPTI-CHEK® blood culture bottle, a Becton Dickinson BACTEC® vial,a Plus Aerobic/F* and Plus Anaerobic/F* culture vial, a Becton DickinsonBACTEC® Standard/10 Aerobic/F culture vial, a Becton Dickinson BACTEC®Myco/F Lytic culture vial, a Becton Dickinson BACTEC® PEDS PLUS/Fculture vial, or a Becton Dickinson BACTEC® Standard Anaerobic/F culturevial (Becton Dickinson, Franklin Lakes, N.J.).

5.2 Exemplary Apparatus

FIG. 1 details an apparatus 11 for determining whether a culture in avessel contains a plurality of microorganisms that comprises a processorand a memory, coupled to the processor. The processor and memoryillustrated in FIG. 1 can be, for example, part of an automated orsemiautomated radiometric or nonradiometric microorganism culturesystem. The apparatus 11 preferably comprises:

-   -   a central processing unit 22;    -   optionally, a main non-volatile storage unit 14, for example a        hard disk drive, for storing software and data, the storage unit        14 controlled by storage controller 12;    -   a system memory 36, preferably high speed random-access memory        (RAM), for storing system control programs, data, and        application programs, comprising programs and data (optionally        loaded from non-volatile storage unit 14); system memory 36 may        also include read-only memory (ROM);    -   a user interface 32, comprising one or more input devices (e.g.,        keyboard 28, a mouse) and a display 26 or other output device;    -   a sensor 34 for taking a measurement of a biological state of a        culture in a vessel;    -   a network interface card 20 (communications circuitry) for        connecting to the sensor 34;    -   an internal bus 30 for interconnecting the aforementioned        elements of the system; and    -   a power source 24 to power the aforementioned elements.

Operation of central processing unit 22 is controlled primarily byoperating system 40. Operating system 40 can be stored in system memory36. In a typical implementation, system memory 36 also includes:

-   -   a file system 42 for controlling access to the various files and        data structures used by the present invention;    -   a microorganism detection module 44 for determining whether a        culture in a vessel contains a plurality of microorganisms;    -   a biological data structure 46 for storing an initial biological        state 48 of the culture and a plurality of measurements of the        biological state of the culture, where each measurement 50 in        the plurality of measurements is taken at a different time point        between a first (initial) time point and a second (final) time        point;    -   an optional lookup table 54 that comprises matches between (i) a        plurality of sets of values, each set of values 56 in the        plurality of sets of values comprising a first threshold value        57 and a second threshold value 58, and (ii) a set of media        types, wherein, for each set of values 56 in the plurality of        sets of values there is corresponding media type 59 in the set        of media types;    -   sets of rate transformation values 60, where each set of rate        transformation values comprises a plurality of rate        transformation values 62, where each rate transformation value        62 is a first derivative of the normalization relative values        associated with a predetermined fixed interval of time points;    -   an average relative transformation value 66 for each set 60 of        rate transformation values 60; and    -   a data structure for storing a determination 68 of whether a        culture in a vessel contains a plurality of microorganisms.

As illustrated in FIG. 1, apparatus 11 can comprise data such asbiological state data structure 46, optional lookup table 54, sets ofrate transformation values 60, average relative transformation values66, and a determination 68 of whether a culture in a vessel contains aplurality of microorganisms. In some embodiments, memory 36 or optionaldata store 14 also stores a measure of central tendency of the averagerelative transformation values 66. The data described above can be inany form of data storage including, but not limited to, a flat file, arelational database (SQL), or an on-line analytical processing (OLAP)database (MDX and/or variants thereof). In some embodiments, such datastructures are stored in a database that comprises a star schema that isnot stored as a cube but has dimension tables that define hierarchy.Still further, in some embodiments, such data structures are stored in adatabase that has hierarchy that is not explicitly broken out in theunderlying database or database schema (e.g., dimension tables that arenot hierarchically arranged). In some embodiments, such data structuresare stored in apparatus 11. In other embodiments, all or a portion ofthese data structures are hosted on (stored on) one or more computersthat are addressable by apparatus 11 across an Internet/network that isnot depicted in FIG. 1. In some embodiments, all or a portion of one ormore of the program modules depicted in apparatus 11 of FIG. 1, such asmicroorganism detection module 44 are, in fact, resident on a device(e.g., computer) other than apparatus 11 that is addressable byapparatus 11 across an Internet/network that is not depicted in FIG. 1.

Apparatus 11 determines the metabolic activity of a culture by, forexample, CO₂ concentration, O₂ concentration, pH, a rate of change inCO₂ concentration, a rate of change in O₂ concentration, or a rate ofchange in pH in a culture. From this metabolic activity determination,apparatus 11 can identify a microorganism type in the culture. In someembodiments, apparatus 11 accommodates a number of culture vessels andserves as an incubator, agitator, and detection system. These componentsof apparatus 11 are not depicted in FIG. 1 because the nature of suchcomponents will vary widely depending on the exact configuration ofapparatus 11. For instance, the number of vessels accommodated byapparatus can range from one vessel to more than 1000 vessels. There canbe a sensor associated with each vessel in order to measure thebiological state of the culture contained within the vessel. The sensorcan be on any location of the vessel and there are a wide range ofpossible sensors that can be used.

FIG. 2 illustrates one exemplary sensor that is capable of measuring thebiological state of a culture. In FIG. 2, a CO₂ sensor 204 is bonded tothe base of culture bottle 202 (vessel) and overlaid with an amount ofculture. CO₂ sensor 204 is impermeable to ions, medium components, andculture but is freely permeable to CO₂. Carbon dioxide produced by thecells in the culture diffuses into sensor 204 and dissolves in the waterpresent in the sensor matrix, generating hydrogen ions. Increases inhydrogen ion concentration (decreases in pH) increase the fluorescenceoutput of sensor 204, thereby changing the signal transmitted fromexcitation filter 206 to emission filter 208. Apparatus 11 takesrepeated measurements of the signal penetrating emission filter 208 overtime and uses this data to determine whether the culture containsmicroorganisms using the algorithms disclosed herein.

In some embodiments, apparatus 11 is an incubator, shaker, andfluorescence detector that will hold between 1 and 1000 culture vessels(e.g., 96, 240 or 384 culture vessels). In some embodiments, the vesselsare arranged in racks (e.g., circular or linear racks), each of whichhas a number of vessel stations. For example, in one specificembodiment, apparatus 11 will hold 240 vessels arranged in six racks,where each rack has 40 vessel stations. In some embodiments, each vesselstation in apparatus 11 contains a light-emitting diode and a photodiode detector with appropriate excitation and emission filters (e.g.,as illustrated in FIG. 2). In some embodiments, the vessels are rockedand heated at 35±1° C.

5.3 Exemplary Method

Now that an exemplary apparatus in accordance with the present inventionhas been described, exemplary methods in accordance with the presentinvention will be detailed. In some embodiments, such methods can beimplemented by microorganism detection module 44 of FIG. 1. Referring tostep 302 of FIG. 3, an initial biological state of the culture is taken.For example, referring to FIG. 2, in some embodiments, an initial readof detector 204 is made to determine the CO₂ concentration in thesensor. In alternative embodiments, an initial O₂ concentration, pH orother indicia of the biological state of the culture is read in step302. In some embodiments, the initial biological state of the culture isdetermined by a fluorescence output of a sensor (e.g., sensor 204) thatis in contact with the culture. In some embodiments, the amount offluorescence output of the sensor is affected by CO₂ concentration inthe manner described above in conjunction with FIG. 2. In someembodiments, the amount of fluorescence output of the sensor is affectedby O₂ concentration, pH, or some other indicia of metabolic state knownin the art. In general, any physical observable that is indicative ofthe metabolic rate of the culture can be measured and stored as theinitial state. In some embodiments, this physical observable is theaccumulation of molecular products (an example being lipopolysaccharidewith Gram negative bacteria), non-molecular physical/chemical changes tothe environment related to growth (pressure changes), and/or theproduction of carbon dioxide or other metabolites that accumulate or theconsumption of substrate such as oxygen) or the accumulation of cellmaterial.

In some embodiments, an initial biological state of the blood culture istaken in step 302 using colorimetric means, fluorometric means,nephelometric means, or infrared means. Examples of colorimetric meansinclude, but are not limited to, the use of the colorimetric redoxindicators such as resazurine/methylene blue or tetrazolium chloride, orthe of p-iodonitrotetrazolium violet compound as disclosed in U.S. Pat.No. 6,617,127 which is hereby incorporated by reference herein in itsentirety. Another example of colorimetric means includes the colormetricassay used in Oberoi et al. 2004, “Comparison of rapid colorimetricmethod with conventional method in the isolation of Mycobacteriumtuberculosis,” Indian J Med Microbiol 22:44-46, which is herebyincorporated by reference herein in its entirety. In Oberoi et al., aMB/Bact240 system (Organon Teknika) is loaded with culture vessels. Theworking principle of this system is based on mycobacterial growthdetection by a colorimetric sensor. If the organisms are present, CO₂ isproduced as the organism metabolizes the substrate glycerol. The colorof the gas permeable sensor at the bottom of each culture vessel resultsin increase of reflectance in the unit, which is monitored by the systemusing infrared rays. Examples of colorimetric means further include anymonitoring of the change in a sensor composition color due to a changein gas composition, such as CO₂ concentration, in a vessel resultingfrom microorganism metabolism.

Examples of fluorometric and colorimetric means are disclosed in U.S.Pat. No. 6,096,272, which is hereby incorporated by reference herein inits entirety, which discloses an instrument system in which a rotatingcarousel is provided for incubation and indexing, and in which there aremultiple light sources each emitting different wavelength light forcolorimetric and fluorometric detection. As used herein nephelometricmeans refers to the measurement of culture turbidity using anephelometer. A nephelometer is an instrument for measuring suspendedparticulates in a liquid or gas colloid. It does so by employing a lightbeam (source beam) and a light detector set to one side (usually 90°) ofthe source beam. Particle density is then a function of the lightreflected into the detector from the particles. To some extent, how muchlight reflects for a given density of particles is dependent uponproperties of the particles such as their shape, color, andreflectivity. Therefore, establishing a working correlation betweenturbidity and suspended solids (a more useful, but typically moredifficult quantification of particulates) must be establishedindependently for each situation.

As used herein, an infrared means for measuring a biological state of ablood culture is any infrared microorganism detection system or methodknown in the art including, but not limited to, those disclosed U.S.Pat. No. 4,889,992, as well as PCT publication number WO/2006071800,each of which is hereby incorporated by reference herein in itsentirety.

In some embodiments the data collected in step 302 and certainsubsequent steps is sorted and collected into a database that includesidentifying information for the vessels such as the identification ofthe vessel (e.g., by sequence and accession numbers), a record of thedates of inoculation, an amount of a biological sample in the culture.

In some embodiments, the vessel 202 holding the culture comprises asensor composition 204 in fluid communication with the culture. In suchembodiments, the sensor composition 204 comprises a luminescent compoundthat exhibits a change in luminescent property, when irradiated withlight containing wavelengths that cause the luminescent compound toluminesce, upon exposure to oxygen. The presence of the sensorcomposition 204 is non-destructive to the blood culture. In suchembodiments, the measuring step 302 (and each instance of the measuringstep 308) comprises irradiating the sensor composition 202 with lightcontaining wavelengths that cause the luminescent compound to luminesceand observing the luminescent light intensity from the luminescentcompound while irradiating the sensor composition with the light. Insome embodiments, the luminescent compound is contained within a matrixthat is relatively impermeable to water and non-gaseous solutes, butwhich has a high permeability to oxygen. In some embodiments, the matrixcomprises rubber or plastic. More details of sensors in accordance withthis embodiment of the present invention are disclosed in U.S. Pat. No.6,900,030 which is hereby incorporated by reference herein in itsentirety.

In optional step 304, the measured initial biological state of theculture upon initialization from step 302 is standardized and stored asthe initial biological state of the blood culture 48 (e.g. to onehundred percent or some other predetermined value). This initialbiological state, stored as data element 48 in FIG. 1, serves as areference value against subsequent measurements of the biological stateof the blood culture. In some embodiments, step 304 is not performed andthe absolute measurements of step 302 are used in the algorithmsdisclosed herein.

Apparatus 11 incubates the culture for a predetermined period of timeafter the initial biological state measurement is taken. Then, after thepredetermined period of time has elapsed, apparatus 11 makes anothermeasurement of the biological state of the culture. This process isillustrated by steps 306 and 308 in FIG. 3. In FIG. 3A, the process isshown as advancing to time step tin step 306. The biological stateduring the time period in step 306 in which apparatus waits for time toadvance by time step t is not used in subsequent processing steps toascertain the microorganism type in the culture. In step 308, once timehas advanced by time step t, a measurement of the biological state ofthe culture in the vessel is again taken in the same manner that theinitial measurement of the biological state was taken (e.g., using thedevice depicted in FIG. 2). In some embodiments, the predeterminedperiod of time (the duration of time step t) is ten minutes. In someembodiments, the predetermined period of time (the duration of time stept) is a period of time that is less than 5 minutes, a period of timethat is less than 10 minutes, a period of time that is less than 15minutes, a period of time that is less than 20 minutes, a period of timein the range between 1 minute and 30 minutes, a period of time in therange between 1 minute and 20 minutes, a period of time in the rangebetween 5 minute and 15 minutes, or a period of time that is greaterthan 5 minutes. The measurement of the biological state of the culturein the vessel taken in step 308 is converted to a normalization relativevalue by standardizing the measurement of step 308 against the initialmeasurement of step 302 in embodiments where the initial measurement ofstep 302 is used for normalization. In one embodiment, the measurementof the biological state of the culture in the vessel taken in step 308is converted to a normalization relative value by taking the ratio ofthe measurement of step 308 against the initial measurement of step 302.In some embodiments, this computed normalization relative value isstored as a data element 50 in FIG. 1. In some embodiments, themeasurement of the biological state measured in step 308 is stored as adata element 50 in FIG. 1 and the normalization relative valuecorresponding to the measurement of the biological state measured instep 308 is computed as needed in subsequent processing steps.

In step 310 a determination is made as to whether a first predeterminedfixed time interval has elapsed. For example, in some embodiments thepredetermined fixed time interval is seventy minutes. In such anexample, if the time step t of step 306 is 10 minutes, then it wouldrequire time step t to have advanced seven times before condition310—Yes is achieved. In some embodiments, the predetermined fixedinterval of time is a duration of time that is between five minutes andfive hours, a duration of time that is between twenty minutes and tenhours, a duration of time that is between twenty minutes and two hours,a duration of time that is between thirty minutes and ninety minutes, aduration of time that is less than 24 hours, or a duration of time thatis more than 24 hours. When the first predetermined fixed interval oftime has elapsed (310—Yes), process control passes on to step 312 wherean additional step of the algorithm is performed. When the firstpredetermined fixed interval of time has not elapsed (310—No), processcontrol passes back to step 306 where the algorithm waits for time toadvance by the amount of time t prior to once again taking a measurementof the biological state of the culture in a new instance of step 308.

The net result of steps 306 through 310 is that a plurality ofmeasurements of a biological state of the culture in the vessel aretaken and that each measurement in the plurality of measurements is at adifferent time point between a first (initial) time point and aterminating (final) time point. Further, in typical embodiments wheretime step t is the same amount in each instance of step 306, themeasurements in the plurality of measurements are each taken of theculture at a periodic interval. In some embodiments, the periodicinterval is an amount of time between one minute and twenty minutes, anamount of time between five minutes and fifteen minutes, an amount oftime between thirty seconds and five hours, or an amount of time that isgreater than one minute.

When a predetermined fixed interval has elapsed (310—Yes), a firstderivative of the normalization relative values in the respectivepredetermined fixed interval (or absolute values from step 302 in therespective predetermined fixed interval in embodiments in whichnormalization is not performed) is computed in step 312, thereby forminga rate transformation value 62. In other words, the change in thenormalization relative values during the predetermined fixed interval isdetermined in step 312. Note that rate transformation values are thefirst derivative of normalization relative values in embodiments wheremeasurement data is normalized and rate transformation values are thefirst derivative of absolute measurements from step 302 in embodimentswhere measurement data is not normalized. In some embodiments, thepredetermined fixed interval of time over which the first derivative iscomputed is all measurements in an immediately preceding period of timethat is between twenty minutes and two hours. For example, in someembodiments the predetermined fixed interval of time of step 310 isseventy minutes and, in step 312, the rate of change across all of thenormalization relative values of measurements in this seventy minutetime interval (the past 70 minutes) is determined in step 312 and storedas the rate transformation value 62. In some embodiments, thepredetermined fixed interval of time over which the first derivative iscomputed (time window) is all measurements in an immediately precedingperiod of time that is between five minutes and twenty hours, betweenthirty minutes and ten hours, between twenty minutes and two hours,between twenty minutes and ten hours, or between thirty minutes andninety minutes.

In step 314 a determination is made as to whether a predetermined numberof rate transformation values have been measured since the last timecondition 314—Yes was reached. If so (314—Yes), process control passeson to step 316. If not (314—No), process control returns back to step306 where process control waits until time step t has elapsed beforecontinuing to step 308 where the normalization relative value of theculture is once again calculated. Each instance of condition (314—Yes)marks the completion of a set 60 of rate transformation values 62. Forexample, in some embodiments, condition 314—Yes is achieved when sevennew rate transformation values 62 have been measured. In this example, aset 60 of rate transformation values comprises or consists of the sevenrate transformation values 62. In some embodiments, each set 60 of ratetransformation values 62 comprise or consists of between four and twentycontiguous rate transformation values. Contiguous rate transformationvalues 62 are rate transformation values in the same set 60. Such ratetransformation values 62 are, for example, calculated and stored insuccessive instances of step 312. In some embodiments, each set 60 ofrate transformation values 62 in the plurality of rate transformationvalues comprises or consists of between five and fifteen contiguous ratetransformation values 62, between one and one hundred contiguous ratetransformation values 62, between five and one fifteen contiguous ratetransformation values 62, more than five rate transformation values 62,or less than ten rate transformation values 62.

When condition 314—Yes is achieved, step 316 is run. In step 316, anaverage relative transformation (average rate of change) value 66 iscomputed from the newly formed set 60 of rate transformation values 62.Thus, for each set 60 of rate transformation values 62, there is anaverage relative transformation value 66. In some embodiments, anaverage relative transformation (average rate of change) value 66 iscomputed from the newly formed set 60 of rate transformation values 62by taking a measure of central tendency of the rate transformationvalues 62 in the newly formed set 60 of rate transformation values 62.In some embodiments, this measure of central tendency is a geometricmean, an arithmetic mean, a median, or a mode of all or a portion of therate transformation values 62 in the newly formed set 60 of ratetransformation values 62.

In step 318, a determination is made as to whether a predetermined pointin the protocol has been reached. This predetermined point is a finaltime point, also known as an end point. In some embodiments, the finaltime point is reached (318—Yes) one or more hours, two or more hours,ten or more hours, between three hours and one hundred hours, or lessthan twenty hours after the initial measurement in step 302 was taken.In some embodiments, the final time point is reached (318—Yes) whenbetween 10 and 50,000, between 100 and 10,000, or 150 and 5,000, morethan 10, more than fifty, or more than 100 measurements of thebiological state of the culture in the vessel have been made ininstances of step 308. If the predetermined point in the protocol hasnot been reached (318—No), then process control returns to step 306where the process control waits for time step t to advance beforeinitiating another instance of step 308 in which the biological state ofthe culture is again measured and used to calculate a normalizationrelative value. If the predetermined point in the protocol has beenreached (318—Yes), process control passes to either (i) step 320 a, (ii)step 320 b, or (iii) both step 320 a and step 320 b.

In step 320 a, a first result is obtained based on a determination ofwhether any average relative transformation value 66 in the plurality ofaverage relative transformation values exceeds first threshold value 57.In some embodiments, the first threshold value 57 is media typedependent meaning that the exact value for the first threshold valuewill depend on the media type that was used for the culture. Inpractice, for example, optional lookup table 54 may store severaldifferent first threshold values 57 for several different media types59. Thus, in step 320 a, the optional lookup table 54 is consulted,based on the exact media type 59 of the culture, to determine thecorrect first threshold value 57 to use. In some embodiments, it isexpected that, regardless of the exact media type 59 used, the firstthreshold value will be in the range of between 50 and 200. In someembodiments, it is expected that, regardless of the exact media type 59used, the first threshold value will be in the range of between 75 and125. In some embodiments, it is expected that, regardless of the exactmedia type 59 used, the first threshold value will be in the range ofbetween 85 and 115. In some embodiments, it is expected that, regardlessof the exact media type 59 used, the first threshold value will be inthe range of between 95 and 105. If any average relative transformationvalue 66 in the plurality of average relative transformation valuesexceeds first threshold value 57 (320 a—Yes), then the culture is markedpositive for microbial infection (step 322). If none of the averagerelative transformation values 66 in the plurality of average relativetransformation values exceeds the first threshold value 57 (320 a—No),then the culture is not marked positive for microbial infection (step324). However, in some embodiments, even if the condition 320 a—No isachieved, other microbial detection algorithms in apparatus 11 may markthe culture as positive for microbial infection. For instance, in someembodiments, step 320 b is run and step 320 b is capable of marking theculture positive for microbial infection. Furthermore, other additionalmicrobial detection algorithms may be run by apparatus 11 on theculture, for example an algorithm that detects an inflection point inthe rate of acceleration of a signal from the culture, and these otheradditional microbial detection algorithms may independently determinethat the culture is infected with a microorganism.

In step 320 b, a second result is obtained based on a determination ofwhether the extent of growth exhibited by the culture exceeds secondthreshold value 58. In some embodiments, the second threshold value 58is media type dependent meaning that the exact value for the secondthreshold value will depend on the media type that was used for theculture. In practice, for example, lookup table 54 may store severaldifferent second threshold values 58 for several different media types59. Thus, in step 320 a, the lookup table 54 is consulted, based on theexact media type 59 of the culture, to determine the correct secondthreshold value 57 to use. If the extent of growth exceeds the secondthreshold value 58 (320 b—Yes), then the culture is marked positive formicrobial infection (step 322). If the extent of growth does not exceedthe second threshold value 58, then the culture is not marked positivefor microbial infection (step 324). However, in some embodiments, evenif the condition 320 b—No is achieved, other microbial detectionalgorithms in apparatus 11 may mark the culture as positive formicrobial infection. For instance, in some embodiments, step 320 a isrun and step 320 a is capable of marking the culture positive formicrobial infection as described above. Furthermore, other additionalmicrobial detection algorithms may be run by apparatus 11 on theculture, for example an algorithm that detects an inflection point inthe rate of acceleration of a signal from the culture, and these otheradditional microbial detection algorithms may independently determinethat the culture is infected with a microorganism.

In some embodiments, the extent of growth (EG) 58 is the maximumnormalization relative value measured for the culture. In someembodiments where EG is the maximum normalization relative value, it isexpected that, regardless of the exact media type 59 used, the secondthreshold value will be in the range of between 112 and 140. In someembodiments where EG is the maximum normalization relative value, it isexpected that, regardless of the exact media type 59 used, the secondthreshold value will be in the range of between 113 and 118. In someembodiments where EG is the maximum normalization relative value, it isexpected that the second threshold value will be 117.

In some embodiments, the extent of growth is determined by the equation:

EG=NR_(after_growth)−NR_(minimum_growth)  Eq. 1

where NR_(after_growth) is a normalization relative value in theplurality of normalization relative values that was used in thecalculation of (i) the first average relative transformation valuefollowing a maximum average relative transformation value, (ii) amaximum average relative transformation value, or (iii) a first averagerelative transformation value preceding the maximum average relativetransformation value in the plurality of average relative transformationvalues, and NR_(minimum_growth) is a normalization relative value in theplurality of normalization relative values that was used in thecalculation of the first average relative transformation value toachieve a third threshold value. In some embodiments where EG is definedby Equation 1, the second threshold value will be in the range ofbetween 12 and 40 regardless of the exact media type 59 used. In someembodiments where EG is defined by Equation 1, the second thresholdvalue will be in the range of between 13 and 18 regardless of the exactmedia type 59 used. In some embodiments where EG is defined by Equation1, the second threshold value will be 17 regardless of the exact mediatype 59 used.

In some embodiments, NR_(after_growth) of Eq. 1 is a normalizationrelative value in the plurality of normalization relative values thatwas used in the calculation of the average relative transformation value66 following the maximum average relative transformation value 66 everachieved for the culture. Thus, if normalization relative values 145through 154 were used to compute the average relative transformationvalue 66 following the maximum average relative transformation value 66,then NR_(after_growth) would be one of the normalization relative valuesin the set of normalization relative values {145, . . . , 154}.

In some embodiments, NR_(after_growth) of Eq. 1 is a normalizationrelative value in the plurality of normalization relative values thatwas used in the calculation of the average relative transformation value66 preceding the maximum average relative transformation value 66 everachieved for the culture. Thus, if normalization relative values 125through 134 were used to compute the average relative transformationvalue 66 immediately preceding the maximum average relativetransformation value 66, then NR_(after_growth) would be one of thenormalization relative values in the set of normalization relativevalues {125, . . . , 134}.

In some embodiments, NR_(after_growth) of Eq. 1 is a measure of centraltendency of all or a portion of the normalization relative values thatwere used in the calculation of the maximum average relativetransformation value 66 ever achieved for the culture. Thus, ifnormalization relative values 135 through 144 were used to compute themaximum average relative transformation value 66, then NR_(after_growth)would be a measure of central tendency (geometric mean, an arithmeticmean, a median, or a mode) of all or a portion of the normalizationrelative values in the set of normalization relative values {135, . . ., 144}.

In some embodiments, NR_(after_growth) of Eq. 1 is a measure of centraltendency of all or a portion of the normalization relative values thatwere used in the calculation of the average relative transformationvalue 66 following the maximum average relative transformation value 66ever achieved for the culture. Thus, if normalization relative values145 through 154 were used to compute the average relative transformationvalue 66 following the maximum average relative transformation value 66,then NR_(after_growth) would be a measure of central tendency (geometricmean, an arithmetic mean, a median, or a mode) of all or a portion ofthe normalization relative values in the set of normalization relativevalues {145, . . . , 154}.

In some embodiments, NR_(after_growth) of Eq. 1 is a measure of centraltendency of all or a portion of the normalization relative values thatwere used in the calculation of the average relative transformationvalue 66 preceding the maximum average relative transformation value 66ever achieved for the culture. Thus, if normalization relative values125 through 134 were used to compute the average relative transformationvalue 66 immediately preceding the maximum average relativetransformation value 66, then NR_(after_growth) would be a measure ofcentral tendency (geometric mean, an arithmetic mean, a median, or amode) of all or a portion of the normalization relative values in theset of normalization relative values {125, . . . , 134}.

In some embodiments, NR_(minimum_growth) is a normalization relativevalue in the plurality of normalization relative values that was used inthe calculation of the first average relative transformation value 66 toachieve a threshold value. Thus, if normalization relative values 20through 29 were used to compute the first average relativetransformation value 66 to achieve a threshold value, thenNR_(minimum_growth) would be one of the normalization relative values inthe set of normalization relative values {20, . . . , 29}.

In some embodiments, NR_(minimum_growth) is a measure of centraltendency of the normalization relative values that were used in thecalculation of the first average relative transformation value 66 toachieve a threshold value. Thus, if normalization relative values 20through 29 were used to compute the first average relativetransformation value 66 to achieve a threshold value, thenNR_(minimum_growth) would be all or a portion of the normalizationrelative values in the set of normalization relative values {20, . . . ,29}.

In some embodiments, where Equation 1 is used to calculate extent ofgrowth 58, the threshold value is, in nonlimiting examples, a valuebetween 5 and 100, a value between 25 and 75, a value between 1 and1000, or a value that is less than 50.

In some embodiments, the extent of growth is determined by the equation:

EG=NR_(max)−NR_(initial)  Eq. 2

where NR_(max) is the maximum normalization relative value in theplurality of normalization relative values and NR_(initial) is a valueof the initial biological state of the culture against which eachnormalization relative value has been standardized against. In someembodiments where EG is defined by Equation 2, the second thresholdvalue will be in the range of between 12 and 40 regardless of the exactmedia type 59 used. In some embodiments where EG is defined by Equation2, the second threshold value will be in the range of between 13 and 18regardless of the exact media type 59 used. In some embodiments where EGis defined by Equation 2, the second threshold value will be 17regardless of the exact media type 59 used.

It will be appreciated that equations 1 and 2 can contain additionalmathematical operations, both linear and nonlinear, and still be used tocompute the extent of growth 58.

In some embodiments, the culture is identified as containingmicroorganisms when it contains (i) a bacterium in theEnterobacteriaceae family or (ii) a bacterium not in theEnterobacteriaceae family. In some embodiments, the culture isidentified as containing microorganisms when it contains (i)Enterobacteriacea, (ii) Staphylococcaceae, (iii) Streptococcus, or (iv)Acinetobacter. In some embodiments, the culture is identified ascontaining microorganisms when it contains Alishewanella, Alterococcus,Aquamonas, Aranicola, Arsenophonus, Azotivirga, Blochmannia, Brenneria,Buchnera, Budvicia, Buttiauxella, Cedecea, Citrobacter, Dickeya,Edwardsiella, Enterobacter, Escherichia, Ewingella, Griimontella,Hafnia, Klebsiella, Kluyvera, Leclercia, Leminorella, Moellerella,Morganella, Obesumbacterium, Pantoea, Pectobacterium, Candidatusphlomobacter, Photorhabdus, Plesiomonas, Pragia, Proteus, Providencia,Rahnella, Raoultella, Salmonella, Samsonia, Serratia, Shigella, Sodalis,Tatumella, Trabulsiella, Wigglesworthia, Xenorhabdus, Yersinia, orYokenella.

In some embodiments, the culture is identified as containingmicroorganisms when it contains Staphylococcus aureus, Staphylococcuscaprae, Staphylococcus epidermidis, Staphylococcus haemolyticus,Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcuspettenkoferi, Staphylococcus saprophyticus, Staphylococcus warneri, orStaphylococcus xylosus bacteria. In some embodiments, the culture isidentified as containing microorganisms when it contains S. agalactiae,S. bovis, S. mutans, S. pneumoniae, S. pyogenes, S. salivarius, S.sanguinis, S. suis, Streptococcus viridans, or Streptococcus uberis.

In some embodiments, the culture is identified as containingmicroorganisms when it contains aerobic bacteria. In some embodiments,the culture is identified as containing microorganisms when it containsanaerobic bacteria.

In some embodiments, the method further comprises outputting the firstresult (the yes or no condition reached in step 320 a), the secondresult (the yes or no condition reached in step 32 b), or adetermination of whether the culture in the vessel contains theplurality of microorganisms to a user interface device (e.g., 32), amonitor (e.g., 26), a computer-readable storage medium (e.g., 14 or 36),a computer-readable memory (e.g., 14 or 36), or a local or remotecomputer system. In some embodiments the first result, the secondresult, or the determination of whether the culture in the vesselcontains the plurality of microorganisms is displayed. As used herein,the term local computer system means a computer system that is directlyconnected to apparatus 11. As used herein, the term remote computersystem means a computer system that is connected to apparatus 11 by anetwork such as the Internet.

5.4 Exemplary Computer Program Products and Computers

The present invention can be implemented as a computer program productthat comprises a computer program mechanism embedded in acomputer-readable storage medium. Further, any of the methods of thepresent invention can be implemented in one or more computers. Furtherstill, any of the methods of the present invention can be implemented inone or more computer program products. Some embodiments of the presentinvention provide a computer program product that encodes any or all ofthe methods disclosed herein. Such methods can be stored on a CD-ROM,DVD, magnetic disk storage product, or any other computer-readable dataor program storage product. Such methods can also be embedded inpermanent storage, such as ROM, one or more programmable chips, or oneor more application specific integrated circuits (ASICs). Such permanentstorage can be localized in a server, 802.11 access point, 802.11wireless bridge/station, repeater, router, mobile phone, or otherelectronic devices. Such methods encoded in the computer program productcan also be distributed electronically, via the Internet or otherwise.

Some embodiments of the present invention provide a computer programproduct that contains any or all of the program modules and datastructures shown in FIG. 1. These program modules can be stored on aCD-ROM, DVD, magnetic disk storage product, or any othercomputer-readable data or program storage product. The program modulescan also be embedded in permanent storage, such as ROM, one or moreprogrammable chips, or one or more application specific integratedcircuits (ASICs). Such permanent storage can be localized in a server,802.11 access point, 802.11 wireless bridge/station, repeater, router,mobile phone, or other electronic devices. The software modules in thecomputer program product can also be distributed electronically, via theInternet or otherwise.

5.5 Kits

Some embodiments of the invention may also comprise a kit to perform anyof the methods described herein. In a non-limiting example, vessels,culture for a sample, additional agents, and software for performing anycombination of the methods disclosed herein may be comprised in a kit.The kits will thus comprise one or more of these reagents in suitablecontainer means.

The components of the kits, other than the software, vessels, and theradiometric or nonradiometric system, may be packaged either in aqueousmedia or in lyophilized form. The suitable container means of the kitswill generally include at least one vial, test tube, flask, bottle,syringe or other container means, into which a component may be placed,and preferably, suitably aliquoted. Where there is more than onecomponent in the kit, the kit also will generally contain a second,third or other additional container into which the additional componentsmay be separately placed. However, various combinations of componentsmay be in a vial. The kits of the present invention also will typicallyinclude a means for containing the reagent containers in closeconfinement for commercial sale. Such containers may include injectionor blow-molded plastic containers into which the desired vials areretained.

6 EXAMPLE

A method was developed that allows an increased confidence level in thenotification of vessel positive status in a blood culture system. Themethod set forth herein exemplifies use of this method in the BACTEC®Blood culture system. The BACTEC® Blood culture system uses fluorescentsensors that report changes to the system when microbial metabolismoccurs. Algorithms are then applied to the sequence of signal data thatare designed to recognize signal changes with time that are indicativeof the presence of growing microorganisms. The user is notified when thesystem recognizes evidence of growth (status change to a positive vial)and the vessel is then processed to confirm the presence of an organism(Gram stain and subculture to a plated medium) before initiatingprocesses to begin organism identification and antimicrobialsusceptibility determinations. The prior art system reports a status ofpresumptive positive as the system has no way of quantifying confidencein the positive determination. The present invention described hereutilized the difference in rate of metabolic change and extent of changeto provide information about the confidence in a positive status changeon an individual vessel basis. The inventive data transformation can beapplied to metabolic or cell growth data in a way that providesconfidence in the positive status of a vessel and essentially eliminatesthe potential for false negative determinations as they currently existin blood culture systems.

Data that was collected with the BACTEC® blood culture system is used asan example of the application of the inventive data transformationillustrated in FIG. 3 and provided examples of the utility of thepresent invention. The BACTEC® system, as described above, usesfluorescent sensors to monitor the changes in metabolic activity withinthe culture through a stream of compensated fluorescence signal datathat is collected at ten minute intervals from a sensor located insidethe culture reagent. The data used in this example was collected fromthe BACTEC® instruments used either in internal seeded culture studiesor collected during a clinical evaluation of the system. The data wassorted and collected into a database at Becton Dickinson that includesthe identification of the vessel (by sequence and accession numbers), arecord of the dates of inoculation, the amount of blood in the sample(it is a blood culture system) and the result with the identification ofthe microorganism found in the vessel (the organism identification wasprovided by the clinical site in the case of the external data). Thealgorithm illustrated in FIG. 3 was applied subsequently for analysis.The utility of this information is for determining whether a culture ina vessel contains a plurality of microorganisms

The inventive data transformations began with an initial normalizationof the vessel signal to a specific output (its initial state uponentering the system), as described above in conjunction with steps 302and 304 of FIG. 3. All subsequent data was represented as a percentageof that initial signal, which has been standardized to 100 percent inthese analyses, as outlined in steps 306 and 308 of FIG. 3. Datameasurements normalized by the initial signal were termed normalizationrelative values. Under ideal theoretical conditions, a normalizationrelative value of 125 means that microorganism metabolism caused thefluorescence measured by the BACTEC® sensor to increase by 25 percentrelative to the initial measurement. The next value that was computedwas the first derivative of the NR value as it changes with time asoutlined in steps 310 and 312 of FIG. 3. This value was the ratetransformation (RT) value and the base RT value used in this exampleuses a periodicity limit of 70 minutes. Any given RT value representedthe rate of percentage change of fluorescence signal over the seventyminutes prior to its calculation. The next value that was computed wasthe ART or average rate change value as outlined in steps 314 and 316 ofFIG. 3. This was calculated as the average of the previous 7 averagerate change value (ART) values that had been calculated and acted as asmoothing function of the RT value.

Examples of the parameters that were computed to determine whether aculture was infected with microorganisms are presented in FIGS. 4, 5 and6. An Escherichia coli culture was analyzed using these quantitativemetrics (the normalized relative values 50, the rate transformationvalues 62, and the average relative transformation values 66). Theculture contained three milliliters of human blood from a subject andwas inoculated with a suspension of E. coli (55 CFU) and entered into aBACTEC® 9000 instrument. The identifier 4942 uniquely identifies theculture that is reported in FIGS. 4, 5, and 6 and can be used to linkthe data to a research and development BACTEC® database. FIG. 4 shows aplot of normalization relative values over time. The vessel was enteredinto the instrument and temperature affects related to equilibration ofthe vessel were observed for approximately the first hour. The signalstabilized and a background was observed to increase from 94 percent to95 percent of the initial signal for the first hour (this rate was dueto blood activity). In the normalization relative plot (FIG. 4), growthwas visible beginning at eight hours and proceeded until 15 hours with afinal value NR value near 126. The plot of average relativetransformation values 66 over time based on the average rate of changein rate transformation values 62 of FIG. 4 over time is provided in FIG.5. Each average relative transformation (ART) value 66 is a measure ofthe average rate of change and the maximum ART for this culture was 1158achieved at 12.8 hours into the culture. This represents this culture'saveraged maximum achieved rate of sensor change over a one hour period.FIG. 6 is the second derivative plot of normalization relative values 50and shows the changes in rate with time. This is a graphicalinterpretation showing the following critical points: the point ofinitial acceleration 602 (movement from the null), the point of maximumacceleration 604 (the maxima), where acceleration reaches its maximum(crossing the null point), the maximum point of deceleration (theminima) 606, and the terminus of the growth curve 608 (where the ratechange returns to null).

The BACTEC® system applies a series of algorithms to the signal datathat can trigger the system to identify a vessel with a positive statusbased on the occurrence of a “knee” or the presence of a change in rateor acceleration in the sequence of data. There is no attempt to qualifythis determination to add a confidence to this status change. Confidencecan be added to the prior art detection algorithm by counting the numberof times a set of algorithms are triggered for a vessel in protocol. Themore times an algorithm was triggered to indicate a positive status thenthe more confidence in the change in status to positive. Although thiswould certainly increase confidence in the result for many cultures itis based on an indicator method that is limited in its ability toadequately and robustly provide the confidence desired in anydetermination in a diagnostic system. As an example, in the externalclinical evaluation of the modified Aerobic plus medium a false negativeculture was observed when using conventional microorganism detectionalgorithms rather than the algorithms disclosed in the presentinvention. The culture was found to contain Enterococcus faecalis whensubcultured at the end of protocol. The data was inspected and it wasdetermined that two door opening events (at 7.0 and 7.9 hours intoprotocol), where the door to the BACTEC® system was literally opened(with concomitant temperature transient events that affected thetemperature), compensated signal data and caused the prior art detectionalgorithms to fail. Applying the ART transformation to the data wouldhave allowed robust detection (possibly delayed by as much as 2 hours).In addition, by applying a threshold based on the ART data(conservatively and an ART value of 100 or greater as positive) thisvessel would be detected by the system on every subsequent reading forup to fourteen hours in protocol (instrument positive on every testcycle for as long as 5 hours). The use of both the ART and the NRtransformation (with positive threshold) could have extended the periodof positive detection possibly to the end of protocol. The point beingthat the use of these transformation provides a very high degree ofconfidence that a vessel is positive, even vessels that have detected asfalse negative on the current system. FIGS. 7, 8 and 9 show data for aculture that was found to contain Enterococcus faecalis when subculturedat the end of protocol. The data in these figures establish that theinventive methods disclosed herein also detect this microorganisminfection. FIG. 7 is a plot of compensated fluorescent signal versustime for a clinical Enterococcus faecalis false negative. FIG. 8 is aplot of normalized relative values versus time for a clinicalEnterococcus faecalis false negative. FIG. 9 is a plot of average ratetransformation versus time for a clinical Enterococcus faecalis falsenegative. Using the methods of the present invention, the culture wouldhave been found to contain microorganisms.

7 REFERENCES CITED

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety herein for all purposes.

8 MODIFICATIONS

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1-55. (canceled)
 56. A method comprising: (A) incubating, with anincubator, a culture disposed within a vessel during portions of a timeinterval between a first time point and a second time point, wherein theculture comprises a sample and a culture media; (B) measuring, with asensor, a biological state of the culture at a plurality of time pointsbetween the first time point and the second time point, wherein thebiological state is one of CO₂ concentration, O₂ concentration, pH, arate of change in CO₂ concentration, a rate of change in O₂concentration, or a rate of change in pH; (C) determining, with one ormore processors, a plurality of rate transformation values, wherein eachrate transformation value is derived from a different subset of themeasurements of the biological state of the culture within apredetermined time interval; (D) determining, with the one or moreprocessors, a plurality of measures of central tendency, wherein eachmeasure of central tendency is derived from a different subset of therate transformation values; (E) determining, with the one or moreprocessors, whether the culture contains a plurality of microorganismsbased on (i) whether at least one measure of central tendency is greaterthan or less than a first threshold value or (ii) whether an extent ofgrowth exhibited by the culture is greater than or less than a secondthreshold value, wherein the extent of growth is derived from one ormore of the measurements of the biological state of the culture; and (F)processing the vessel to confirm the presence of the plurality ofmicroorganisms if it is determined in step (E) that the culture containsa plurality of microorganisms.
 57. The method of claim 56, whereinprocessing the vessel to confirm the presence of the plurality ofmicroorganisms in step (F) comprises at least one of (i) preparing agram stain or (ii) preparing a subculture on a plated medium.
 58. Themethod of claim 56 further comprising: initiating processes formicroorganism identification and antimicrobial susceptibility testingwhen it is confirmed in step (F) that the culture contains a pluralityof microorganisms.
 59. The method of claim 56 further comprising:displaying the determination of step (E) on a user interface device. 60.The method of claim 56, wherein the sensor used in step (B) to measurethe biological state of the culture at the plurality of time points is acolorimetric sensor.
 61. The method of claim 56, wherein the sensor usedin step (B) to measure the biological state of the culture at theplurality of time points is a fluorometric sensor.
 62. The method ofclaim 56, wherein the sensor used in step (B) to measure the biologicalstate of the culture at the plurality of time points is a nephelometricsensor.
 63. The method of claim 56, wherein the sensor used in step (B)to measure the biological state of the culture at the plurality of timepoints is an infrared sensor.
 64. The method of claim 56, wherein thedetermination of step (E) is based on the comparison between the atleast one measure of central tendency and the first threshold value. 65.The method of claim 64, wherein each rate transformation valuedetermined in step (C) is a first derivative of a different subset ofthe measurements of the biological state of the culture within apredetermined time interval.
 66. The method of claim 64 furthercomprising: converting the measurements of the biological state of theculture into normalization relative values, wherein each ratetransformation value determined in step (C) is a first derivative of adifferent subset of the normalization relative values within apredetermined time interval.
 67. The method of claim 64, wherein eachmeasure of central tendency determined in step (D) is a geometric meanof a different subset of the rate transformation values.
 68. The methodof claim 64, wherein each measure of central tendency determined in step(D) is an arithmetic mean of a different subset of the ratetransformation values.
 69. The method of claim 64, wherein each measureof central tendency determined in step (D) is a median of a differentsubset of the rate transformation values.
 70. The method of claim 64,wherein each measure of central tendency determined in step (D) is amode of a different subset of the rate transformation values.
 71. Themethod of claim 64, wherein the rate transformation values arecalculated and stored in successive instances, and wherein the measuresof central tendency are calculated and stored in successive instances.72. The method of claim 71, wherein all of the predetermined timeintervals have the same duration.
 73. The method of claim 56, whereinthe determination of step (E) is based on the comparison between theextent of growth exhibited by the culture and the second thresholdvalue.
 74. The method of claim 73 further comprising: converting themeasurements of the biological state of the culture into normalizationrelative values, wherein each rate transformation value determined instep (C) is a first derivative of a different subset of thenormalization relative values within a predetermined time interval. 75.The method of claim 74, wherein the rate transformation values arecalculated and stored in successive instances, and wherein the measuresof central tendency are calculated and stored in successive instances.76. The method of claim 75, wherein all of the predetermined timeintervals have the same duration.
 77. The method of claim 75, whereinthe extent of growth is a maximum normalization relative value measuredfor the culture.
 78. The method of claim 75, wherein the extent ofgrowth (EG) is determined by the equation:EG=NR_(after_growth)−NR_(minimum_growth), wherein: NR_(after_growth) isa normalization relative value in the plurality of normalizationrelative values that was used in the calculation of (i) a maximummeasure of central tendency in the plurality of measures of centraltendency determined in step (D), (ii) a first measure of centraltendency following the maximum measure of central tendency, or (iii) afirst measure of central tendency preceding the maximum measure ofcentral tendency; and NR_(minimum_growth) is a normalization relativevalue in the plurality of normalization relative values that was used inthe calculation of a first measure of central tendency.
 79. The methodof claim 75, wherein the extent of growth (EG) is determined by theequation:EG=NR_(after_growth)−NR_(minimum_growth), wherein: NR_(after_growth) isa normalization relative value in the plurality of normalizationrelative values that was used in the calculation of a measure of centraltendency following a maximum measure of central tendency in theplurality of measures of central tendency determined in step (D); andNR_(minimum_growth) is a normalization relative value in the pluralityof normalization relative values that was used in the calculation of afirst measure of central tendency.
 80. The method of claim 75, whereinthe extent of growth (EG) is determined by the equation:EG=NR_(after_growth)−NR_(minimum_growth), wherein: NR_(after_growth) isa normalization relative value in the plurality of normalizationrelative values that was used in the calculation of a measure of centraltendency preceding a maximum measure of central tendency in theplurality of measures of central tendency determined in step (D); andNR_(minimum_growth) is a normalization relative value in the pluralityof normalization relative values that was used in the calculation of afirst measure of central tendency.
 81. The method of claim 75, whereinthe extent of growth (EG) is determined by the equation:EG=NR_(after_growth)−NR_(minimum_growth), wherein: NR_(after_growth) isa measure of central tendency of all or a portion of the normalizationrelative values that were used in the calculation of a maximum measureof central tendency in the plurality of measures of central tendencydetermined in step (D); and NR_(minimum_growth) is a measure of centraltendency of the normalization relative values that were used in thecalculation of a first measure of central tendency.
 82. The method ofclaim 75, wherein the extent of growth (EG) is determined by theequation:EG=NR_(after_growth)−NR_(minimum_growth), wherein: NR_(after_growth) isa measure of central tendency of all or a portion of the normalizationrelative values that were used in the calculation of a measure ofcentral tendency following a maximum measure of central tendency in theplurality of measures of central tendency determined in step (D); andNR_(minimum_growth) is a measure of central tendency of thenormalization relative values that were used in the calculation of afirst measure of central tendency.
 83. The method of claim 75, whereinthe extent of growth (EG) is determined by the equation:EG=NR_(after_growth)−NR_(minimum_growth), wherein: NR_(after_growth) isa measure of central tendency of all or a portion of the normalizationrelative values that were used in the calculation of a measure ofcentral tendency preceding a maximum measure of central tendency in theplurality of measures of central tendency determined in step (D); andNR_(minimum_growth) is a measure of central tendency of thenormalization relative values that were used in the calculation of afirst measure of central tendency.
 84. The method of claim 75, whereinthe extent of growth (EG) is determined by the equation:EG=NR_(max)−NR_(initial), wherein: NR_(max) is a maximum normalizationrelative value in the plurality of normalization relative values; andNR_(initial) is a value of an initial biological state of the cultureagainst which each normalization relative value has been standardizedagainst.
 85. The method of claim 56, wherein the determination of step(E) is based on (i) the comparison between the at least one measure ofcentral tendency and the first threshold value and (ii) the comparisonbetween the extent of growth exhibited by the culture and the secondthreshold value.
 86. The method of claim 56, wherein it is determined instep (E) that the culture contains a plurality of microorganisms, andwherein the plurality of microorganisms is aerobic.
 87. The method ofclaim 56, wherein it is determined in step (E) that the culture containsa plurality of microorganisms, and wherein the plurality ofmicroorganisms is anaerobic.